The eucaryotic cell cycle is regulated by cyclin dependent kinases (CDKs). These are phosphoenzymes (kinases), which are only activated after binding with a cyclin subunit, and do not have the activity by themselves. Various processes in cell cycles, such as replication and introduction to mitosis, are regulated by different CDKs. The expression levels of CDKs are generally consistent and the activities of CDKs are dependent on the expression levels of cyclins. Cyclins are transiently expressed at required stages in the cell cycle and activate CDKs.
Additionally, CDK activity is also regulated by a group of inhibitory factors, called CDK inhibitors. These inhibitors are roughly classified into two groups based their primary structure and their specificity for inhibiting CDK, particularly, the INK4 family (Inhibitor of CDK4), and the CIP/KIP family (CDK interacting protein/kinase inhibitory protein). Comparing the progression of the cell cycle to driving an automobile, CDKs play a role of accelerator, and CDK inhibitors play the role of a brake. The progression of cell cycle is determined by the concerted regulation of both of the accelerator and the brake.
When quiescent cells enter the cell cycle, D-type cyclins are expressed from the middle to late G1 phase in response to the mitogenic stimulation. Ras/Raf-1/MAPK accelerate the transcription of the genes coding for D-type cyclins, and PI3K (phosphatidylinositol 3-kinase)/Akt (protein kinase B) repress the degradation of the gene products. CDK4 and CDK6 bind to D-type cyclins, the assembled proteins enter the cell nucleus where they must be phosphorylated by CAK (CDK-activating kinase) to become active. D-type cyclins have been reported to be a cytoplasmic sensor for stimulation signals, i.e., their expression is induced in response to extracellular growth factors, and they play a role for transferring the signal to CDK2 and CDC2 which progress the cell cycle. It is also reported that D-type cyclin/CDK4, 6 have two roles for cell cycle progression. One is the role of canceling the growth inhibitory effect of RB (the retinoblastoma protein) through phosphorylation and another is trapping (sequestering) CIP/KIP. When CIP/KIP exists alone in the cell, the activity of CDK2 appeared in G1 anaphase, has been inhibited. It is speculated that D-type cyclin/CDK reduces the activity of such inhibitors as CIP/KIP to inhibit CDK2 by associating therewith.
In G1 phase of cell cycle, the target of CDK is RB. RB is known as to associate with many proteins, especially with a key molecule, transcription factor E2F. E2F regulates the transcription of the genes which are necessary for progression of the cell cycle and DNA replication. For example, it activates the transcription of cyclin E. Thus, E2F plays an important role for initiation of the S phase through the function of cyclin E/CDK2. The non-phosphorylated RB binds with E2F strongly, and represses the DNA replication by E2F. When RB is phosphorylated CDK, the phosphorylated RB loses the function to repress E2F, and becomes inactive. [Recently, it has been reported that the homologous proteins of RB and E2F. They are called the RB family and the E2F family respectively.] As a result, it has been revealed that the progression of the cell cycle from G1 to the S phase is a highly regulated process by both the RB and E2F family proteins. Further, this RB-E2F pathway was revealed to be related to many biological events such as cell differentiation, malignant transformation and apoptosis. For example, inactivation of RB and the like causes the abnormal regulation of cell cycle, and leads to the malignant transformation of the cell. A number of these genes, which regulate the cell cycle, are reported to be cancer suppressor genes.
On the other hand, it is known that terminal differentiated cells such as cardiomyocytes and nerve cells withdraw from the cell cycle and take on the special state called the stationary phase (G0 phase). Cardiomyocytes, one of these terminal differentiated cells loses proliferative activity soon after birth. Thus, the necrosis or loss of cardiomyocytes by infarction or dilated cardiomyopathy leads to failure of regeneration of cardiomyocytes. Thereafter, there are a lot of cases for severe heart failure and death resulting in a high mortality rate of these cardiac diseases. Although it has been thought that cardiomyocytes are arrested at G0 phase and the cell cycle does not proceed, recent studies suggest that cardiomyocytes have the regulatory mechanisms of the cell cycles.
For example, adenovirus transforming gene product E1A promotes transcription of E2F dependent genes by interacting with the RB, and induces cellular DNA synthesis. A study of whether E1A releases E2F in cardiomyocytes and induces DNA synthesis and dedifferentiation using rat neonatal cultured cardiomyocytes showed E1A alone did not clearly induce the DNA synthesis but leads to apoptosis. In the presence of E1B, E1A is reported to induce DNA synthesis in cardiomyocytes (Kirshenbaum, L. A. et al., The Journal of Biological Chemistry 270, 7791-7794 (1995)).
As a result of using E2F adenovirus, it has been reported that E2F represses the expression of myocardial specific genes in cardiomyocytes, and induces DNA synthesis (Kirshenbaum, L. A. et al., Developmental Biology 179, 402-411, (1996)).
On the other hand, the transgenic mouse, in which a wild type cyclin D1 without nuclear localization signals was overexpressed, showed elevated expression levels of CDK4, as well as, DNA synthesis in cardiomyocytes, however, it was reported that abnormal multinucleated cells were increased (Soonpaa, M. H et al., Clin. Invest. 99, 2644-2654, (1997)).
From such a line of research, DNA synthesis is observed and progression of cell cycles is suggested in cardiomyocytes, however, subsequent cell division or increase of cell numbers has not been reported. Thus, any method for substantially proliferating cardiomyocytes without induction of apoptosis is not known.
Recently, for the purposes of organ transplantation or treatment of leukemia and the like, the regeneration medical technology to produce desired kinds of cells using multipotent cells called as embryonic stem (ES) cells have been studied. However, because ES cells are produced by disrupting an embryo that has a potential to grow into a fetus, there are many ethical resistances to the research.
In view of the above problems, the invention is directed a method for proliferating terminal differentiated cells, which is applicable for the development of cardiac regenerating therapy and self-transplantation of well-differentiated organs such as cardiomyocytes. It is a further object of the invention to provide a recombinant vector that can be used for above method, or a pharmaceutical composition thereof.